Lipoxygenase

ABSTRACT

The invention provides sequence information of a microbial protein having lipoxy-genase activity and a method of producing the protein by recombinant DNA technology. More specifically, the inventors have isolated a gene encoding a lipoxygenase from  Gaeu - mannomyces graminis , cloned it into an  E. coli  strain and sequenced it. A comparison shows less than 25% identity to known lipoxygenase sequences, the closest being human 15S li-poxygenase. The inventors have expressed the lipoxygenase recombinantly and found that the recombinant lipoxygenase is glycosylated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK01/00574 filed Sep. 5, 2001 (the international application waspublished under PCT Article 21(2) in English) and claims priority or thebenefit under 35 U.S.C. 119 of Danish application nos. PA 2000 01320 andPA 2001 00322 filed Sep. 5, 2000 and Feb. 27, 2001, respectively, andSwedish application no. 0004790-2 filed on Dec. 22, 2000 and U.S.provisional application No. 60/272,604 filed Mar. 1, 2001, the contentsof which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a polynucleotide encoding alipoxygenase and its use for recombinant production of a lipoxygenase.The invention also relates to a method of obtaining a lipoxygenase byscreening a DNA library with specific probes.

BACKGROUND OF THE INVENTION

Lipoxygenase is an enzyme that catalyzes the oxygenation of linoleicacid and produces a hydroperoxide. It is classified in EnzymeNomenclature as EC 1.13.11.12. The enzyme is widely distributed inplants and animals. Encoding genes have been isolated from varioussources, and the sequences have been published. Thus, GENESEQP W93832and Genbank U78294 give the sequence of human 15S lipoxygenase.

Microbial lipoxygenases are known from a yeast Saccharomyces cerevisiae,a thermophilic actinomycete Thermoactinomyces vulgaris, from fungusFusarium oxysporum, Fusarium proliferatum and Gaeumannomyces graminis(Su and Oliw, J. Biological Chemistry, 273 (21), 13072-13079 (1998)). Noisolated gene encoding a microbial lipoxygenase has been described.

The prior art describes various uses of lipoxygenase, e.g. as a foodadditive to bread dough or noodles.

SUMMARY OF THE INVENTION

Here we for the first time provide sequence information of a microbialprotein having lipoxygenase activity and a method of producing theprotein in industrial scale. More specifically, the inventors haveisolated a gene encoding a lipoxygenase from Gaeumannomyces graminis,cloned it into an E. coli strain and sequenced it. The genome of G.graminis contains approximately 60% of the G and C nucleotides, whichmade this work very difficult. A comparison shows less than 25% identityto known lipoxygenase sequences, the closest being human 15Slipoxygenase. The inventors have expressed the lipoxygenaserecombinantly.

Accordingly, the invention provides a polypeptide having lipoxygenaseenzyme activity which:

a) has an amino acid sequence which has at least 50% identity with themature polypeptide of SEQ ID NO: 2 or 23;

b) is encoded by a nucleic acid sequence which hybridizes at 55° C. witha complementary strand of the nucleic acid sequence encoding the maturepolypeptide of SEQ ID NO: 1 or a subsequence thereof having at least 100nucleotides;

c) has an amino acid sequence which can be obtained from the maturepoly-peptide of SEQ ID NO: 2 or 23 by substitution, deletion, and/orinsertion of one or more amino acids; or

d) is encoded by the lipoxygenase-encoding part of the DNA sequencecloned into a plasmid present in Escherichia coli deposit number DSM13586.

The invention also provides a polynucleotide which comprises:

a) the partial DNA sequence encoding a mature lipoxygenase cloned into aplasmid present in Escherichia coli DSM 13586,

b) the partial DNA sequence encoding a mature lipoxygenase shown in SEQID NO: 2 or 23,

c) an analogue of the sequence defined in a) or b) which encodes alipoxygenase and

i) has at least 50% identity with said DNA sequence, or

ii) hybridizes at low stringency with a complementary strand of said DNAsequence or a subsequence thereof having at least 100 nucleotides,

iii) is an allelic variant thereof, or

d) a complementary strand of a), b) or c).

Other aspects of the invention provide a nucleic acid constructcomprising the polynucleotide, a recombinant expression vectorcomprising the nucleic acid construct, and a recombinant host celltransformed with the nucleic acid construct. The invention also providesa recombinant method of producing the lipoxygenase, an oligonucleotideprobe based on SEQ ID NO: 2 or 23 and a method of obtaining alipoxygenase by screening a eukaryotic DNA library using the probe basedon SEQ ID NO: 2.

Further, the invention provides a dough composition comprising amanganese lipoxygenase and a method for preparing a dough or a bakedproduct made from dough, comprising adding a manganese lipoxygenase tothe dough. The invention also provides a method of oxygenating asubstrate selected from the group consisting of linolenic acid,arachidonic acid, linoleyl alcohol and a linoleic acid ester comprisingcontacting the substrate in the presence of oxygen with a manganeselipoxygenase. Finally, the invention provides a detergent compositioncomprising a manganese lipoxygenase and a surfactant.

DETAILED DESCRIPTION OF THE INVENTION

Genomic DNA Source

DNA encoding the lipoxygenase (LOX) may be derived from fungi,particularly Ascomycota,more particularly Ascomycota incertae sedis e.g.Magnaporthaceae, such as Gaeumannomyces, or anamorphic Magnaporthaceaesuch as Pyricularia, or alternatively anamorphic Ascomycota such asGeotrichum. An example is G. graminis, e.g. G. graminis var. graminis,G. graminis var. avenae or G. graminis var. tritici, particularly thestrain G. graminis var. graminis CBS 903.73, G. graminis var. avenae CBS870.73 or G. graminis var. tritici CBS 905.73. The CBS strains arecommercially available from Centraalbureau voor Schimmelcultures, Baarn,the Netherlands.

The inventors obtained two LOX-encoding DNA sequences from strains ofGaeumannomyces graminis and found that they have the sequences shown inSEQ ID NO: 1 and 22. They inserted a LOX-encoding gene into a strain ofEscherichia coli and deposited it as E. coli DSM 13586 on 5 Jul. 2000under the terms of the Budapest Treaty with the DSMZ—Deutsche Sammlungvon Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig DE, Germany. The deposit was made by Novo Nordisk A/S andwas later assigned to Novozymes A/S.

Lipoxygenase

The lipoxygenase of the invention is a manganese lipoxygenase, i.e. ithas lipoxygenase activity (EC 1.13.11.12) with manganese in theprosthetic group. It is glycosylated and may have a molecular weight inthe range 90-110 kDa, particularly 95-105 kDa. It is thermostable with atemperature optimum of 65-90° C., particularly 75-85° C. Thelipoxygenase is stable against LAS (linear alkyl-benzene sulfonate) upto 400 ppm at pH 10. Mn-Lipoxygenase is enzymatically active between pH5-12 with a broad optimum at pH 6-8.

A recombinant lipoxygenase may have a higher glycosylation and a higherthermostability. The recombinant lipoxygenase may have a molecularweight in the range 90-110 kDa, particularly 95-105 kDa. It may have atemperature optimum of 65-90° C., particularly 75-85° C.

Recombinant Expression Vector

The expression vector of the invention typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a selectable marker, atranscription terminator, a repressor gene or various activator genes.The vector may be an autonomously replicating vector, or it may beintegrated into the host cell genome.

Production by Cultivation of Transformant

The lipoxygenase of the invention may be produced by transforming asuitable host cell with a DNA sequence encoding the lipoxygenase,cultivating the transformed organism under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture.

The host organism may be a eukaryotic cell, in particular a fungal cell,such as a yeast cell or a filamentous fungal cell, e.g. a strain ofAspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A.niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S.cerevisiae. The production of the lipoxygenase in such host organismsmay be done by the general methods described in EP 238,023 (NovoNordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

Nucleotide Probe

A nucleotide probe may be designed on the basis of the DNA sequence ofSEQ ID NO: 1 or the polypeptide sequence of SEQ ID NO: 2, particularlythe mature peptide part. The probe may be used in screening forLOX-encoding DNA as described below.

A synthetic oligonucleotide primer may be prepared by standardtechniques (e,g, as described in Sambrook J, Fritsch E F, Maniatis T(1989) Molecular cloning: a laboratory manual (2^(nd) edn.) Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) on the basis of the maturepart of the amino acid sequence in SEQ ID NO: 2 or the correspondingpart of the DNA sequence. It may be a degenerate probe and willtypically contain at least 20 nucleotides.

Screening of Eukaryotic DNA Library

A polypeptide with lipoxygenase activity may be obtained by a methodcomprising:

a) preparing a eukaryotic DNA library,

b) screening the library to select DNA molecules which hybridize to theprobe described above,

c) transforming host cells with the selected DNA molecules,

d) cultivating the transformed host cells to express polypeptidesencoded by the DNA molecules, and

e) assaying the expressed polypeptides to select polypeptides havinglipoxygenase activity.

The eukaryotic DNA library may be prepared by conventional methods. Itmay include genomic DNA or double-stranded cDNA derived from suitablesources such as those described above.

Molecular screening for DNA sequences may be carried out by polymerasechain reaction (PCR) followed by hybridization.

In accordance with well-known procedures, the PCR fragment generated inthe molecular screening may be isolated and subcloned into a suitablevector. The PCR fragment may be used for screening DNA libraries by e.g.colony or plaque hybridization.

Hybridization

The hybridization is used to indicate that a given DNA sequence isanalogous to a nucleotide probe corresponding to a DNA sequence of theinvention. The hybridization may be done at low, medium or highstringency. One example of hybridization conditions is described indetail below.

Suitable conditions for determining hybridization between a nucleotideprobe and a homologous DNA or RNA sequence involves presoaking of thefilter containing the DNA fragments or RNA in 5×SSC (standard salinecitrate) for 10 min, and prehybridization of the filter in a solution of5×SSC (Sambrook et al. 1989), 5× Denhardt's solution (Sambrook et al.1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA(Sambrook et al. 1989), followed by hybridization in the same solutioncontaining a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)Anal. Biochem. 132:6-13), ³²P-dCTP-labeled (specific activity>1×10⁹cpm/μg) probe for 12 hours at approx. 45□ C. The filter is then washedtwo times for 30 minutes in 2×SSC, 0.5% SDS at a temperature of at least55□ C, particularly at least 60□ C, more particularly at least 65□ C,e.g. at least 70□ C, or at least 75□ C

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an x-ray film.

Alignment and Identity

The nucleotide sequence of the invention may have an identity to thedisclosed sequence of at least 75% or at least 85%, particularly atleast 90% or at least 95%, e.g. at least 98%.

For purposes of the present invention, alignments of sequences andcalculation of identity scores were done using a Needleman-Wunschalignment (i.e. global alignment), useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R.Pearson and D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA”, Methods inEnzymology, 183:63-98).

Use of Lipoxygenase

A manganese lipoxygenase such as that described above may be used in thefollowing application, e.g. in analogy with the indicated publications.

The lipoxygenase can be used as an additive to dough for baked productssuch as bread, biscuits and cakes. Thus, the lipoxygenase can be used ina process for making bread, comprising adding the lipoxygenase to adough, kneading the dough and baking the dough to make the bakedproduct. SU 426640 A, JP 58190346 A[SLK1], JP 1165332 A[SLK2], JP8322456,[SLK3] JP 10028516[SLK4], JP 08322456, JP 2964215. It can alsobe used in the preparation of noodles as described in JP 11299440 A.

The lipoxygenase may be used for bleaching, e.g. bleaching ofbeta-carotene, wheat flour or wheat dough. U.S. Pat. Nos.1,957,333-1,957,337.

It can also be used for oxidizing mixtures of fatty acids to hydroperoxyfatty acids, as accelerators of lipid peroxidition, and as analytictools to estimate linoleic and linolenic acids contents of certain oils.

The invention provides a detergent composition comprising thelipoxygenase and a surfactant, particularly an anionic surfactant suchas LAS (linear alkyl-benzene sulfonate). Advantageously, thelipoxygenase has good stability in the presence of such surfactants. Thedetergent may be formulated as described in U.S. Pat. No. 3,635,828[SLK5]or U.S. Pat. No. 5,789,362[SLK6]. The lipoxygenase can also beused to bleach stains from fabrics or hard surfaces as described in DK9800352[SLK7]. Advantageously,

The lipoxygenase can be used for modification of starch as mentioned inJP 09163953, EP772980, JP 2000-106832. Also it can be used for proteinmodification as described in EP 947142, DE 19840069 or JP 61078361, ormodification of oil (production of conjugated fatty acid) as mentionedin JP 5905128, U.S. Pat. No. 3,729,379.

The lipoxygenase can be used for cross-linking a protein by oxidases,such as laccase, bilirubin oxidase etc. EP 947142.

The lipoxygenase can be used to obtain improved glutinousness andimproved flavor of marine paste product such as Kamaboko, Hanpen, byadding lipoxygenase to fish meat. JP 61078361.

The lipoxygenase can be used to produce a process tomato product. It canbe used for tomato pasta, salsa, ketchup and so on. EP 983725.

The lipoxygenase can be used for production of hydroperoxy fatty acid byreacting lipoxygenase with unsaturated 4-24C fatty acid. JP 11029410.

The hydroperoxides of linoleic acid or linolenic acid can be convertedfurther to e.g. growth regulatory hormone jasmonic acid, and the productfrom arachidonic acid can be converted to physiological effectorsleukotrienes and lipoxins.

Application of lipoxygenase should not be limited to the examplesmentioned above. Since hydroperoxide, the product of lipoxygenasereaction, is good oxidant to create radical, lipoxygenase can be usedfor any other applications utilizing oxidation reaction, such asbleaching of food material or textile dyes, or polymerization ofchemical compounds to produce plastic material or fiber.

Assay for Lipoxygenase Activity

The lipoxygenase activity was determined spectrophotometrically at 25°C. by monitoring the formation of hydroperoxides. For the standardanalysis, 10 μL enzyme was added to a 1 mL quartz cuvette containing 980μL 25 mM phosphate buffer (pH 7.0) and 10 μL of substrate solution (10mM linolenic acid dispersed with 0.2%(v/v) Tween20). The enzyme wastypically diluted sufficiently to ensure a turn-over of maximally 10% ofthe added substrate within the first minute. The absorbance at 234 nmwas followed and the rate was estimated from the linear part of thecurve. One unit causes an increase in absorbance at 234 nm of 0.001/min.

Determination of Substrate Specificity

The substrate specificity of the lipoxygenase was studied using thestandard assays condition with a number of different compounds assubstrate. All substrates were produced as dispersions with 0.2%(v/v)Tween20. The amount of compound added to make up these stock solutionswas determined by mass, since viscosity made accurate measurement ofvolume impossible. The limiting rate constant and the specificityconstant were determined by varying the amount of substrate added in theassays. The resulting rates were plotted against the concentration ofsubstrate used. Finally, the plots were fitted by non-linear leastsquares regression to the theoretical hyperbolic curve of theMichaelis-Menten equation. The cis-trans-conjugated hydro(pero)xy fattyacids were assumed to have a molecular extinction coefficient of 23,000M⁻¹ cm⁻¹.

EXAMPLES

Materials and Methods

Molecular cloning techniques are described in Sambrook et al. (1989).

The following commercial plasmids and E. coil strains were used forsub-cloning and DNA library construction:

-   -   pT7Blue (Novagen)    -   pUC19 (TOYOBO, Japan)    -   E. coli JM109 (TOYOBO, Japan)    -   E. coli DH12□ (GIBCO BRL, Life Technologies, USA)

The following commercial Kits were used for cDNA cloning;

-   -   cDNA Synthesis Kit (Takara, Japan)    -   Marathon cDNA Amplification Kit (Clontech, USA)    -   Oligo dT cellulose powder (Invitrogen, Netherlands)

Labeling and detection of hybridization probe was done usingDIG-labeling and detection Kit (Boehringer Manheim). Nylon membraneHybond-N+ (Amersham, England) was used for DNA transfer for bothsouthern blotting and colony hybridization.

Media and Buffer Solution

COVE-ar: per liter 342.3 g sucrose, 20 ml COVE salt solution, 10 mMacrylamide, 15 mM CSCl₂, 30 g Agar noble (Difco)

COVE2-ar: per liter 30 g sucrose, 20 ml COVE salt solution, 10 mMacrylamide, 30 g Agar noble (Difco)

COVE salt solution: per liter 26 g KCl, 26 g MgSO₄-7H₂O, 76 g KH₂PO₄, 50ml Cove trace metals.

Cove trace metals: per liter 0.04 g NaB₄O₇-10H₂O, 0.4 g CuSO₄-5H₂O, 1.2g FeSO₄-7H₂O, 0.7 g MnSO₄-H₂O, 0.7 g Na₂MoO₂-2H₂O, 0.7 g ZnSO₄-7H₂O.

AMG trace metals: per liter 14.3 g ZnSO₄-7H₂O, 2.5 g CuSO₄-5H₂O, 0.5 gNiCl₂, 13.8 g FeSO₄, 8.5 g MnSO₄, 3.0 g citric acid.

YPG: per liter 4 g yeast extract, 1 g KH₂PO₄, 0.5 g MgSO₄-7H₂O, 15 gglucose, pH 6.0.

STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl₂.

STPC: 40% PEG4000 in STC buffer.

Cove top agarose: per liter 342.3 g sucrose, 20 ml COVE salt solution,10 mM Acelamide, 10 g low melt agarose.

MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0.

MDU-2 Bp: per liter 45 g maltose-1H₂O, 7 g yeast extract, 12 g KH₂PO₄, 1g MgSO₄-7H₂O, 2 g K₂SO₄, 5 g Urea, 1 g NaCl, 0.5 ml AMG trace metalsolution pH 5.0.

Materials

alpha-³²P-dCTP (3000 Ci/mmol), dNTPs, alpha-³³P-ddNTPs, Hybond-Nmembranes, and DNA labeling beads (-dCTP), T-primed first-strand kit,and Thermo Sequenase kits were from Amersham Pharmacia Biotech (Uppsala,Sweden). TA cloning kits were from Invitrogen (Groningen, TheNetherlands). Taq DNA polymerase and the enhanced avian RT-PCR kit werefrom Sigma (St. Louis, Mo.). Restriction enzymes were from New EnglandBioLabs (Beverly, Mass.). G. graminis was obtained and grown asdescribed by Su and Oliw (supra). Qiagen plant RNeasy mini and OIAquickgel extraction kits were from Merck Eurolab (Stockholm, Sweden).Degenerate primers for PCR were obtained from TIB Molbiol (Berlin,Germany), whereas sequencing primers were purchased from CyberGene(Huddinge, Sweden). 5′-RACE and reverse transcription of total RNA wasperformed with a kit (5′RACE system for rapid amplification of cDNAends) from Life Technologies (Täby, Sweden).

Example 1 Determination of Partial Peptide Sequences of LOX from G.graminis

A fungal strain of Gaeumannomyces graminis var. tritici was cultivatedand lipoxygenase was recovered essentially as described in Chao Su andErnst H. Oliw, J. Biological Chemistry, 273 (21), 13072-13079 (1998).

To obtain data from the N-terminal part of the enzyme, approximately 10mg of enzyme was analyzed directly by using traditional edmandegradation on the 494 Protein Sequencer, Applied Biosystems accordingto the manufacturer's instructions.

Another 40 microgram of sample was lyophilized down to around 20 μl andadded 20 μl SDS-sample buffer containing DTT before incubation 30 min at37° C. and then boiling the sample for 3 min. 5 μl 0.5 M iodoacetamidein 1 M Tris-HCl, pH 7.5 was then added and the sample was incubated 20min at room temperature prior to running the sample on SDS-PAGE (4-20%,Novex) according to the manufacturer's instructions. The gel was stainedaccording to standard procedures from Novex.

The gelpiece (60 kDa) was subsequently cut out and minced with a blade.The gel pieces were washed 2× in 0.5 M tris pH 9.2/ACN (1:1) for 45 minat 37° C. The gel pieces were treated with 100% ACN for 10 min tointroduce shrinking of the pieces. The ACN was removed and the piecesdries in speed-Vac. 200 ml 0.1 M NH4CO3 (AMBIC) was added and incubatedfor 15 min. AMBIC was removed and 100 ml ACN added. Again incubation for10 min followed by removal of ACN and drying in speed-vac. The cyclewith AMBIC was repeated 2×. After the last drying step 20 ml 0.05 mg/mltrypsin in 0.1 M tris pH 9.2, 10% ACN was added. Incubation for 10 min.Then 300 ml 0.1 M tris pH 9.2, 10% ACN was added. Incubation wascontinued O.N. at 37° C. The supernatant was then removed (saved forcontrol) and the peptides extracted from the gel by adding 30 ml 10%TFA. After 5 min the TFA was withdrawn and collected. Further extractionwas done 2× by adding 150 ml 0.1% TFA, 60% ACN to the gel pieces andincubate for 30 min at 37° C. All extracts were collected (30 ml+150ml+150 ml) and concentrated in the speed-vac to 50 ml. A sample of theconcentrate (5 ml) was run on RP-HPLC on a Vydac C-18 column usingsolvent system of TFA/isopropanol to see if any peptides were present.The rest of the sample was run to collect the peptides. Controls withblank gel pieces were run in parallel. To minimize loss of peptide,selected fractions were sequenced directly without any repurification.

The resulting N-terminal sequence is shown as SEQ ID NO: 21, and twointernal peptides (denoted fr 29 and 34) are shown as SEQ ID NOS: 19 and20.

Further, around 100 μg lipoxygenase was added 40 μl 0.05 M potassiumphosphate, 10 mM EDTA, 1% Triton X-100, 0.05% SDS, pH 7.3 and heated to90° C. for 4 min and allowed to cool. Then the sample was added 25 mUO-glycosidase (BSA free) and 800 mU EndoF glycosidase (Boehringer) andleft over night at 37° C. The sample was then added 75 gl SDS samplebuffer and run on SDS-PAGE (Novex 4-20%) in 7 lanes according to themanufacturer's instructions.

The 60 kDa bands were cut out from the gel minced and washed twice ineppendorf tubes with 400 μl of 0.5 M Tris-HCl, pH 9.2:ACN 1:1 for 45 minat 37° C. The gel pieces were then treated with 200 μl ACN for 10 minand then dried in the speed vac. 400 μl NH4HCO3 was added and left for10 min before removing the supernatant and treating the pieces withanother 200 μl of ACN for 10 min and then drying. 400 μl H2O was addedand the sample left for 10 min before repeating the procedure with ACNagain. The gel pieces was then added 25 μl 0.1 mg/ml trypsin+300 μl 0.1M Tris-HCl, 10% ACN, pH 9.2 and left over night at 37° C. Afterincubation 35 μl of 10 TFA was added and the supernatant were takenafter 30 min for HPLC (Vydac C18, gradient to 80% acetonitril in 0.1%TFA). The gel pieces were then further extracted twice with 150 μl 0.1%TFA, 60% acetonitril. The supernatant was taken and evaporated in thespeed vac to around 50 μl before adding further 100 μl 0.1% TFA and thenre-evaporating down to 50 μl which was then run on the HPLC.

Three amino acid sequences (denoted fr 20, 21 and 25) were obtained, asshown in SEQ ID NOS: 16,17 and 18.

Example 2 Cloning of Genomic and cDNA Clone of LOX From G. graminis

Preparation of Fungal Chromosomal DNA

A fungal strain Gaeumannomyces graminis var. triftici was cultivated inthe YPG (composed per liter: 4 g Yeast extract, 1 g KH₂PO₄, 0.5 g MgSO₄7H₂O, 15 g Glucose, pH 6.0) with gentle agitation at 25° C. for 6 days.Mycelia was collected by filtration using Mira-cloth (Calbiochem, USA)and washed with deionized water twice. After briefly dried on paperfilter, mycelia was frozen by liquid nitrogen and ground by motor on dryice. Around 0.2 g ground mycelia was put into a 1.5 ml eppendorf tubeand suspended in 0.5 ml of buffer solution composed with 100 mM NaCl, 25mM EDTA, 1% SDS and 50 mM Tris-HCl (pH 8). After addition of 3 micro-lof 25 mg/ml proteinase K, the tube was incubated at 65° C. for 30-60minutes. The solution was extracted with the same volume of phenol andDNA was precipitated with 0.7 volume of isopropanol at −20° C. Thepellet was re-suspended in 0.5 ml of sterilized water and remaining RNAwas digested by 50 micro-g of RNase at 37° C. for 30 minutes. DNA wasphenol extracted and ethanol precipitated again. The pellet wasresuspended in appropriate amount of sterilized water.

Preparation of mRNA and Synthesis of cDNA

A fungal strain Gaeumannomyces graminis var. tritici was cultivated inthe YPG with gentle agitation at 25° C. for 6 days. After thelipoxygenase activity was confirmed, mycelia was collected and ground ondry ice as mentioned before to be used for the preparation of total RNAwith phenol-chloroform method. Purification of mRNA from total RNA wasperformed with Oligo dT cellulose powder (Invitrogen, Netherland).

Synthesizing of cDNA was done with cDNA Synthesis Kit (Takara, Japan).The first strand cDNA was synthesized using 5-6 micro-g of heatdenatured mRNA as the template in the mixture containing 1.0 mM each ofdNTP, 4 μg of oligo(dT)_(1B) and 2 μg of random primer and 100 U ofreverse transcriptase and 1^(st) strand synthesis buffer. In total 50 μlof reaction mixture was kept at room temperature for 10 min, thenincubated at 42° C. for 1 hour. After the incubation, the reactionmixture was chilled on ice for 2 min and subjected to 2^(nd) strand cDNAsynthesis. 1138 U of E. coli DNA polymerase and 5 μl of E. coli RNaseH/E. coli DNA ligase mixture and 2^(nd) DNA synthesis buffer was addedto the 1^(st) strand synthesis mixture and diluted up to 240 μl withDEPC-H₂O. The reaction mixture was incubated at 12° C. 1 hour, 22° C. 1hour and 70° C. 10 min. Then 10 U of T4 DNA polymerase was added to thereaction mixture and incubated at 37° C. 10 min. Synthesized cDNA wassubjected to agarose gel electrophoresis to confirm the quality.

Isolation of a Partial Clone of LOX Gene by PCR

The following primers were designed and synthesized based on the aminoacid sequences determined in Example 1. The nucleotide sequence oflinoleate diol synthase of Gaeumannomyces graminis (Genbank Accession #:AF124979) was used as a reference of codon usage.

Primer 1 for N-term side: SEQ ID NO: 9 (corresponding to amino acids 1-5of N-terminal SEQ ID NO: 21).

Primer 2 for C-term side 1: SEQ ID NO: 10 (corresponding to amino acids18-25 of fr 34, SEQ ID NO: 20).

Primer 3 for C-term side 2: SEQ ID NO: 11 (corresponding to amino acids6-15 of fr 34, SEQ ID NO: 20).

Polymerase chain reaction (PCR) was employed using 0.6 μg of chromosomalDNA of G. graminis as the template in 50 micro-I reaction mixturecontaining 2.5 mM each of dNTP, 20 pmol each of primer 1 and 2, 2.5units of LA taq polymerase (Takara, Japan) and GC buffer I supplied byTakara for LA taq. Reaction condition was shown below. LA taq polymerasewas added to the reaction mixture after step 1.

Step Temperature Time 1 98° C. 10 mins 2 96° C. 20 sec 3 53° C. 45 sec 472° C. (27 + 3 × cycle) sec 5 72° C. 10 mins *Step 2 to Step 4 wererepeated 50 times.

Second PCR reaction was employed in the reaction mixture described abovebut using 2 μl of first PCR product as template and primer 3 instead ofprimer 2. Reaction condition was the same as described above except step2 to step 4 were repeated 30 times.

Amplified 1 kb fragment was gel-purified using QIAquick™ Gel ExtractionKit (Qiagen) and subcloned into pT7Blue. Sequence of the PCR clone wasdetermined as shown in SEQ ID NO: 3. From the deduced amino acidsequence of the PCR fragment, the primer 1 turned out to be hybridizedto elsewhere than expected, however, amino acid sequence 250599Bfr25(SEQ ID NO: 18) determined in Example 1 was found in continuous 216amino acids sequence in the PCR fragment (SEQ ID NO: 8). Identity searchshowed that the 216 amino acid sequence had the highest identity toHuman 15S Lipoxygenase (Genbank U78294, GENESEOP W93832), Humanarachidonate 12-Lipoxygenase (Swiss-Prot P18054) and Plexaura homomalla8R-Lipoxygenase (GenBank AF003692, SPTREMBL O16025). The resultsindicated that the obtained PCR fragment contained lipoxygenase gene.The highest score of identity was obtained with Human 15S and was lessthan 25%.

Cloning of Genomic LOX Gene

To obtain a full-length genomic clone, southern blotting was employed ongenomic DNA of G. graminis using PCR fragment as a probe. Based on theresult, genomic DNA was digested with Sall and separated on 1.0% agarosegel. Around 6 kb of DNA digestion was recovered from the gel and ligatedwith BAP treated pUC19 lineared by Sall. Ligation mixture wastransformed into E. coli DH12S to construct a partial genomic library.It was screened by colony hybridization using the PCR fragment as probe,and a positive E. coli colony was isolated and the plasmid, termedpSG16, was recovered. The plasmid pSG16 contained a 6 kb Sall fragmentfrom G. graminis. Out of 6 kb of this fragment, sequence of 4.1 kblength including the PCR clone was determined as shown in SEQ ID NO: 4.The largest open reading frame (ORF) contained the above-mentioned 216amino acid sequence as well as the similar sequences to fr 20, 21, 29and 34, SEQ ID NOS: 16, 17, 19 and 20 but not the N-terminal sequence(SEQ ID NO: 21) determined in example 1. Two other small ORFs were foundin the upstream of the largest ORF, but none of them had the N-terminalsequence neither. To find the right initial ATG codon, cDNA cloning wasnecessary.

Isolation of cDNA Clone of LOX Gene

Total RNA was extracted from the mycelia producing lipoxygenase andsubjected for mRNA preparation by Oligo dT cellulose powder. The cDNAwas synthesized from the mRNA using cDNA Synthesis Kit (Takara, Japan)and aiming to obtain full-length cDNA, 1-4 kb of cDNA was gel-purifiedto be subjected for the construction of a partial cDNA library. Librarywas constructed by ligating with the adaptor of Marathon cDNAAmplification Kit (Clontech, USA), which allows the amplification ofaimed cDNA with the Adaptor Primer (AP1) and a custom primer designedfor the internal sequence of aimed clone.

For the amplification of cDNA of LOX, two primers, primer 4 (SEQ ID NO:12) and primer 5 (SEQ ID NO: 13), were designed based on the sequence ofgenomic clone. C-terminal part was amplified with primer 4 and AP1, andN-terminal part was amplified with primer 5 and AP1.

PCR reaction mixture comprised of 2.5 mM dNTP, 30 pmol each of primer 4and AP1 or primer 5 and AP1, 5 units of LA taq polymerase (Takara) andsupplied GC buffer 1. Reaction condition was shown below. LA taqpolymerase was added to the reaction mixture after step 1.

Step Temperature Time 1 98° C. 5 mins 2 95° C. 30 sec 3 74° C. 15 sec 468° C. 3 mins 5 95° C. 30 sec 6 95° C. 5 mins 7 54° C. 30 sec 8 68° C.15 sec *Step 2 to Step 4 were repeated 15 times and the temperature ofStep 3 was decreased 4° C. after each 3 repeat. Step 6 to Step 8 wererepeated 20 times.

As the results, 0.6 kb and 1.6 kb fragments were amplified for 5′-endand 3′-end respectively and the sequences were determined as shown inSEQ ID NO: 5 and SEQ ID NO: 6. Based on the sequence around thepredicted initial ATG and stop codon TAA, the primer 6 (SEQ ID NO: 14)and primer 7 (SEQ ID NO: 15) were designed for the amplification ofend-to-end cDNA. Also desired restriction enzyme sites were introducedat both ends for further plasmid construction.

Reaction mixture contained 0.08 μg of cDNA library, 2.5 mM dNTP, 30 pmoleach of primer 6 and 7, 1 units of LA taq polymerase (Takara) and GCbuffer. Reaction condition was shown below. LA taq polymerase was addedto the reaction mixture after step 1.

Step Temperature Time 1 98° C. 10 mins 2 96° C. 20 sec 3 53° C. 45 sec 472° C. (27 + 3 × cycle) sec 5 72° C. 10 mins *Step 2 to Step 4 wererepeated 50 times.

PCR amplified 1.9 kb fragment was isolated and cloned into pT7Blueresulting in pSG26. Sequence of the full-length cDNA was determined. Thededuced open reading frame consisted of of 1857 bp, which correspondedto 618 amino acids and a molecular mass of 67600 Da. Comparison with thegenomic sequence turned out that the LOX gene contained one intron inthe N-terminal side. Predicted N-terminal sequence by signal sequencedetermination program is “ALPLAAEDAAAT”. Identity search with thefull-length amino acid sequence showed that it had the highest identityto Human 15S Lipoxygenase (Genbank Accession number w93832), less than25%.

The plasmid pSG26 was transformed in E. coli JM109 and deposited at DSMZas DSM 13586 with the accession date 5 Jul. 2000.

Example 3 Expression of G. graminis LOX in A. oryzae

Host organism

Aspergillus oryzae BECh2 is described in Danish patent application PA1999 01726. It is a mutant of JaL228 (described in WO98/123000), whichis a mutant of IFO4177.

Transformation of A. oryzae

Aspergillus oryzae strain BECh2 was inoculated in 100 ml of YPG mediumand incubated at 32° C. for 16 hours with stirring at 80 rpm. Grownmycelia was collected by filtration followed by washing with 0.6 M KCland re-suspended in 30 ml of 0.6 M KCl containing Glucanex® (NovoNordisk) at the concentration of 30 μl/ml. The mixture was incubated at32° C. with the agitation at 60 rpm until protoplasts were formed. Afterfiltration to remove the remained mycelia, protoplasts were collected bycentrifugation and washed with STC buffer twice. The protoplasts werecounted with a hematitometer and re-suspended in a solution ofSTC:STPC:DMSO (8:2:0.1) to a final concentration of 1.2×10⁷protoplasts/ml. About 4 μg of DNA was added to 100 μl of protoplastsolution, mixed gently and incubated on ice for 30 minutes. 1 μl STPCbuffer was added to the mixture and incubated at 37° C. for another 30minutes. After the addition of 10 ml of Cove top agarose pre-warmed at50° C., the reaction mixture was poured onto COVE-ar agar plates. Theplates were incubated at 32° C. for 5 days.

SDS-PAGE

SDS polyacrylamide electrophoresis was carried out using thecommercialized gel PAGEL AE6000 NPU-7.5L (7.5T%) with the apparatusAE-6400 (Atto, Japan) following the provided protocol. 15 μl of samplewas suspended in 15 μl of 2×conc. of sample loading buffer (100 mMTris-HCl (pH 6.8), 200 mM Dithiothreitol, 4% SDS, 0.2% Bromophenol blueand 20% glycerol) and boiled for 5 minutes. 20 μl of sample solution wasapplied to a polyacrylamide gel, and subjected for electrophoresis inthe running buffer (25 mM Tris, 0.1% SDS, 192 mM Glycine) at 20 mA pergel. Resulting gel was stained with Coomassie brilliant blue.

Construction of Expression Plasmid

The plasmid pSG26 containing cDNA of G. graminis LOX was digested byBg/ll and Xhol and 1.9 kb of fragment which contained the LOX gene wasligated with pMT2188 digested with BamHl and Xhol. The plasmid pMT2188has a modified Aspergillus niger neutral amylase promoter, Aspergillusnidulans TPI leader sequence, Aspergillus niger glucoamylase terminator,Aspergillus nidulans amdS gene as a marker for fungal transformation andS.cerevisiae ura3 as the marker for E.coli transformation.Transformation was done with E. coli DB6507 in which pyrF gene isdeficient and can be complemented with S.cerevisiae Ura3. Resultingplasmid was termed pSG27.

Expression of G. graminis LOX in A. oryzae

A. oryzae BECh2 was transformed with the plasmid pSG27 and selectionpositive transformants were isolated. Transformants were grown on COVE2-ar at 32° C. for 5 days and inoculated to 100 ml of MS-9 shakingflask. After the cultivation with vigorous agitation at 32° C. for 1day, 3 ml of each culture was transferred to 100 ml of MDU-2 Bp inshaking flask to cultivate at 32° C. for 3 days. Culture broth wascentrifuged at 3500 rpm for 10 minutes and supernatant was collected.Lipoxygenase activities of the supernatant were determinedspectrophotometrically as described before. Positive transformantsshowed about 50,000U/ml culture broth while untransformed A. oryzaeBECh2 showed no activity. Culture supernatant was also subjected toSDS-PAGE analysis. Positive transformants showed 90-110 kDa smear bandwhich indicated the protein was heavily glycosylated. UntransformedA.oryzae BECh2 did not show any major band.

Example 4 Purification of Recombinant Lipoxygenase

One gram of crude lyophilised lipoxygenase prepared as in the previousexample was dissolved in 40 mL 25 mM Tris-HCl (pH 8.0) and then filtered(0.45 μm, type Millex-HV, Millipore). The above and subsequent stepswere all carried out at room temperature. The filtrate was loaded on aSP-Sepharose Fast Flow (2.6×14 cm) with 25 mM Tris-HCl (pH 8.0) at 1mL/min. The column was then washed with the same buffer at 2.5 mL/minuntil baseline was reached (approximately 4 column volumes). The boundprotein was then eluted with a linear gradient from 0 to 330 mM NaCl in25 mM Tris-HCl (pH 8.0) in 2 column volumes. Fractions of 10 mL werecollected. The column was cleaned with 1 M NaCl in 25 mM Tris-HCl (pH8.0). The fractions containing the majority of pure lipoxygenase, asestimated by SDS-PAGE and by activity assay, were pooled andconcentrated using an Amicon cell (10,000 NMWL, YM10, Millipore). Theenzyme was finally transferred into 50 mM sodium phosphate (pH 7.0) bydialysis and stored in aliquots at −20° C. until use.

SDS-PAGE analysis showed that the lipoxygenase had been purified tohomogeneity. The enzyme was found to have an estimated molecular weightof 90-110 kDa, somewhat higher than the theoretical value based on theamino acid sequence (65.6 kDa). This was taken as an indication ofglycosylation. The protein was found to have a very high isoelectricpoint as demonstrated by the successful purification employing cationexchange chromatography.

Example 5 Determination of the Gene and the Deduced Protein Sequence ofMn-lipoxygenase

1. Amino Acid Sequences of Internal Peptides and the C-Terminal AminoAcids of Manganese Lipoxygenase

Manganese lipoxygenase was purified to homogeneity as described by Suand Oliw (supra), using a strain of G. graminis (different from theprevious examples). Internal peptides were generated, purified andsequenced by the Sanger method essentially as described for anotherprotein of G. graminis (Hornsten L, Su C, Osbourn A E, Garosi P, HellmanU, Wernstedt C and Oliw E H, Cloning of linoleate diol synthase revealshomology with prostaglandin H synthases. J Biol Chem 274(40): 28219-24,1999). The N-terminal amino acid of Mn-lipoxygenase was blocked, butfour C-terminal amino acid was obtained by C-terminal sequencing.

(i) C Terminal Amino Acid Sequence

These C-terminal amino acids were FLSV.

(ii) Internal Amino Acid Sequences

The following eight internal amino acid sequences were obtained (where(K), (K/R) and (E) denotes the fact that Lys-C, trypsin and V8 cleavespeptides at the C-terminal side of K residues, K or R residues, and Eresidues, respectively):

(K)LYTPQPGRYAAACQGLFYLDARSNQFLPLAIK (amino acids 205-237 of SEQ ID NO:23 with the substitution K206L)

(K/R)HPVMGVLNR (amino acids 295-304 of SEQ ID NO: 23 with Lys or Arg atposition 295)

(K/R)LFLVDHSYQK (amino acids 196-205 of SEQ ID NO: 23 with Lys or Arg atposition 196)

(E)M?AGRGFDGKGLSQG(W/M)PFV (amino acids 569-587 of SEQ ID NO: 23, exceptthat amino acid 570 is uncertain Met and amino acid 584 is Trp or Met)

(K/R)GLVGEDSGPR (amino acids 365-375 of SEQ ID NO: 23 except that aminoacid 365 was found to be Lys or Arg and 368 Val)

(K)TNVGADLTYTPLD/AD/WK/LP/ND/NE (amino acids 237-255 of SEQ ID NO: 23except that amino acid 242 was found to be Ala, 250 Asp or Ala, 251 andAsp or Trp)

(K)G/F SGVLPLHPAw (amino acids 472-483 of SEQ ID NO: 23, except thatamino acid 473 was found to be Gly or Phe, and amino acid 483 uncertainTrp)

(K) QTVDDAFAAPDLLAGNGPGRA (amino acids 532-553 of SEQ ID NO: 23 exceptthat amino acid 536 was found to be Asp, and 552 Arg)

2. RT-PCR with Degenerate Primers Generated cDNA of Mn-lipoxygenase

This part of the invention was difficult due to the high GC content ofthe genome of G. graminis.

Methods for isolation of total RNA from G. graminis and transcription ofmRNA to cDNA had to be optimised. cDNA was often contaminated withgenomic DNA in spite of digestion with DNAses and other precautions.

After considerable experimentation, using over 30 degenerate primers invarious combinations, the first cDNA clone of Mn-lipoxygenase could beobtained by RT-PCR. It was obtained by the following degenerate primers,which were based on internal peptides 1 and 2 and above.

(SEQ ID NO: 25) Mn60 (5′-AACCAGTTCCTSCCSCTCGCSATCAA), (SEQ ID NO: 26)Mn15R (5′-GTCGAGGTAGAAGAGGCCCTGRCAVGC), (SEQ ID NO: 27) EO3a(5′-CATCCSGTSATGGGYGTSCTBAA), (SEQ ID NO: 28) EOr3a(5′-CGGTTSAGGACRCCCATVACVGGRTG).

The primers Mn60 and EOr3A generated an RT-PCR band of about 230-bp andthe primers EO3A and Mn15R generated an RT-PCR band of about 220-bp. Asense primer from this sequence (MnS2: 5′-CCGTTCAGCGTCGAGAGCAAGG (SEQ IDNO: 29)) and an antisense primer from the other sequence (MnS1,5′-TCTCGGGGATCGTGTGGAAGAGCA (SEQ ID NO: 30)) amplified a fragment of337-bp. The amplicon was sequenced and it contained the amino acidsequence of peptide1 in one of the reading frames. The amplicon was usedas probe for Northern blot analysis and for screening of a genomiclibrary (Hornsten et al., supra).

3. Screening of a Genomic Library of G. graminis

A genomic library of G. graminis in Lambda ZAP II was obtained asdescribed by Bowyer P et al., Science 267(5196): 371-4, 1995. It wasscreened with a probe of 0.33-kb from the cDNA sequence. Screening ofover 100 000 plagues yielded 11 positive clones, which were plaguepurified by 2-3 additional rounds of phage screening. The Bluescript SKphagemid was excised with helper phage following published methods.Restriction enzyme analysis showed that all rescued phagemids containedthe same insert of 8-kb.

4. Sequencing of the Gene and Coding Region of Mn-LO of G. graminis

Sequencing was performed of both strands using two different methodsbased on cycle sequencing. The sequencing was difficult due to the highGC content of the gene (over 60% GC).

3.4-kb of the genome of G. graminis was sequenced and the sequence of2725 nucleotides of the Mn-lipoxygenase gene included an intron of133-bp. The gene of Mn-lipoxygenase was identified by 5′-RACE from thestarting point of transcription of 2 mRNA, a¹gcaggttc, and the proteintranslation start point A⁷²TG (at nucleotide position 72). TheC-terminal amino acids FLSV were found with the stop codon at position2060-2062. Over 0.6-kb of the 3′-untranslated region was sequenced andtentative polyadenylation signals were found as shown below:

5-RACE and cDNA sequencing was used to confirm the deduced open readingframe and the exon-intron borders. The transcription start point, thetranslation start point and the translation end were determined as shownin SEQ ID NO: 22 and 23.

The Intron was found to have a length of 133 bp and to have the sequenceshown as SEQ ID NO: 24. It was found to be located between nucleotides372 and 373, i.e. between Ser108 and Arg109 of SEQ ID NO: 22.

Example 6 Expression of Native and Genetically Modified Mn-lipoxygenase

We have subcloned a genomic segment (3-kb) containing the coding regionof the Mn-lipoxygenase gene from the Bluescript SK phagemid into themulti cloning site (with SpeI and NsiI sites) of the plasmid pGEM-5Zf(Promega) using the restriction enzymes SpeI and Nsil.

The 5′-end and the intron were modified as follows. pGEM-5Z with theinsert was cleaved with SpeI and BseRI, which cut out the 5′-end of thegene and part of the genomic sequence with the intron (1323-bp). Thispiece was replaced in pGEM with a cDNA sequence of about 405-pb, whichwas obtained by cleavage of a PCR product of 448-bp with SpeI and BseR1.This vector is designated pGEM_Met. The PCR product was generated with asense primer specific to the translation start region (and with SpeI andNdeI site in the 5′-end of the primer,5′-TTACTAGTCATATGCGCTCCAGGATCCTTGCT (SEQ ID NO: 31)), and a genespecific antisense primer located at the 3′-end of the BseR1 site. ThiscDNA part so inserted thus contained the beginning of the ORF (withoutthe Intron positioned between nucleotides 372 and 373, between Ser108and Arg109, as shown in the table above), so that the entire ORF wasobtained in the vector pGEM_Met.

The 3′-end was modified with PCR, taking advantage of an BbvCI siteabout 130-bp from the stop signal. The sense primer was gene-specificand located at the 5′-side of the restriction site, whereas theantisense primer was designed from the nucleotides of the terminal aminoacids and contained, in addition, NdeI and NsiI restriction sites. ThepGEM_Met vector was cleaved with NsiI and BbvC1, and the excisedfragment was replaced with the PCR product cleaved in the same way. Thisyielded the vector pGEM-Met_ter. The modified coding region ofMn-lipoxygenase in this vector can thus be excised with NdeI. Allmodifications have been confirmed by sequencing of the expressionconstructs.

1. Expression of Mn-Lipoxygenase in Procaryotic Cells (E. coli)

The expression vector pET-19b has been linearized with NdeI, and themodified coding region of Mn-lipoxygenase has been excised with NdeI andligated into this vector for expression in E coli, as suggested by themanufacturer of the pET expression vectors (Stratagene). Studies ofrecombinant Mn-lipoxygenase expressed in E. coli is now in progress.

2. Expression of Mn-Lipoxygenase in Eukaryotic Cells (Pichia pastoris,Saccharomyces cerevisiae, Aspergillus nidulans, Gaeumannomyces graminis)

We plan to use the Pichia Expression kit with the pCIC9 or relatedvectors (Invitrogen), which has to be slightly modified to fit ourmodified coding region of Mn-lipoxygenase. It is possible thatglycosylation of recombinant Mn-lipoxygenase may differ betweendifferent hosts. We therefore plan to investigate a series of eukaryoticexpression systems in Saccharomyces cerevisiae, Aspergillus nidulans,Gaeumannomyces graminis. Glucosylation may improve the stability of therecombinant enzyme.

3. Expression of Mn-Lipoxygenase in Eukaryotic Cells (Insect Cells)

We plan to use the Drosophila Expression System (Schneider 2 cells) fromInvitrogen using an expression vector without His tags at the C-terminalend.

4. Genetically modified Mn-Lipoxygenase for expression.

Our discovery that Mn-lipoxygenase belongs to the lipoxygenase genefamily opens large possibilities for rational modification of thestructure. The 3D sequence of several lipoxygenases are known andMn-lipoxygenase shows significant amino acid identity along manyα-helices of soybean lipoxygenase-1 (Prigge S T, Boyington J C, GaffneyB J and Amzel L M, Structure conservation in lipoxygenases: structuralanalysis of soybean lipoxygenase-1 and modeling of human lipoxygenases.Proteins 24(3): 275-91, 1996), which has been used for modeling of manylipoxygenases. Both the metal ligands and other structurally importantamino acids of Mn-lipoxygenase will be mutated in order to increase thebleaching properties and oxidative properties of the enzyme.

4.1 Site directed mutagenesis of amino acids of important alpha-helices.

Amino acid sequences of Mn-lipoxygenase align with α-helix 9 (Prigge etal., supra), which contains the WLLAK sequence and two His residues,which likely are Mn ligands. Systematic changes of amino acids in thishelix might have profound effect on enzyme activity and bleachingproperties. In the same way, an amino acid sequence of Mn-Lipoxygenasealign with α-helix 18, which contain iron ligands and likely Mn-ligands(His and Asn). Other predicted α-helices of Mn-lipoxygenase, whichshould be mutated, correspond to α-helices 7, 8, 10-17, 19-22 of soybeanlipoxygenase-1 (Prigge et al., supra). We predict that some of thesegenetically modified Mn-lipoxygenases may have totally differentproperties, and the bleaching effect may be enhanced. Predicted Mnligands thus are 3 His residues, one Asp residue and one Val residue.Mn-lipoxygenase likely belongs to enzymes of the “2-His-1-carboxylfacial triad”.

4.2 Site directed mutagenesis of amino acids of the C-terminal end.

We plan to mutate the terminal Val to an Ile or to other residues and todetermine the bleaching properties of the mutated form.

4.3 Mosaic Forms of Mn-lipoxygenase

In order to improve the properties of Mn-lipoxygenase we plan substitutevarious parts with the corresponding sequence of soybean lipoxygenaseusing the α-helix information described above.

Example 7 Screening of Eukaryotic DNA

To screen for homologous lipoxygenase genes in eukaryotic fungalstrains, southern hybridization was performed on the genomic DNA fromseveral fungal strains using cDNA of Gaeumannomyces graminis LOX gene asthe probe. Strains of the following species were tested; Pyriculariaoryzae, Psaliota campestris, Penicillium roqueforti and Geotrichumcandidum ATCC34614. Genomic DNA was isolated as described in Example 2.

The probe was labeled with digoxigenin-dUTP using DIG DNA labeling Mix(Boehringer Mannheim) as follows; DIG labeled probe was prepared by PCRusing primer 6 (SEQ ID NO: 14) and primer 7 (SEQ ID NO: 15) as thefull-length cDNA of G. graminis LOX. PCR reaction mixture contained 0.1μg of pSG26 as the template, 1.25 mM dNTP, 8% DIG DNA Labeling Mix, 30pmol each of primer 6 and 7, 1 unit of LA taq polymerase (Takara) and GCbuffer. Reaction conditions were as shown below. LA taq polymerase wasadded to the reaction mixture after step 1.

Step Temperature Time 1 98° C. 10 mins 2 94° C. 2 mins 3 60° C. 30 sec 472° C. 2 mins 5 72° C. 10 mins *Step 2 to Step 4 were repeated 30 times.

PCR products were gel-purified and denatured by heating at 98° C. beforeuse.

About 5 micro-g of DNA digested with restriction enzyme was separated on1.0% agarose gel and denatured by soaking the gel in 0.2N HCl for 30minutes and in 0.5N NaOH +1.5M NaCl for 30 minutes, then and neutralizedin 1M Tris (pH 7.5)+1.5M NaCl for 30 minutes. Denatured DNA was thentransferred to the nylon membrane by vacuum transfer with 20×SSC for 15minutes. After fixing by UV irradiation, nylon membrane was used for thehybridization. Hybridization solution was composed with 5×SSC, 0.5%blocking reagent (Boehringer Mannheim), 0.1% N-lauroylsarcosine and0.02% SDS. The nylon membrane was prehybridized with the hybridizationsolution at 60° C. for 1 hour. After that, the heat-denaturedDIG-labeled probe was added to the hybridization solution and incubatedat 60° C. overnight. Resulting membrane was washed with washing buffercomprising 2×SSC+0.1% SDS for 5 minutes at room temperature twicefollowed by washing with washing buffer 2 composed with 0.1×SSC+0.1% SDSfor 15 minutes at hybridization temperature twice. Washed membrane wasair-dried and used for the detection of DIG-labeled DNA by following theprovided protocol of DNA detection Kit (Boehringer Mannheim).

As the result, Pyricularia oryzae showed clear positive signals andGeotrichum candidum showed very weak signals. The results indicate thatPyricularia oryzae has a lipoxygenase gene that has a high identity toGaeumannomyces graminis LOX and Geotrichum candidum has a gene that haslow identity to G. graminis LOX.

Example 8 Effect of pH on Mn-Lipoxygenase

The activity of lipoxygenase produced as in Example 4 was tested atvarious pH values. The enzyme was found to have a broad pH optimum withhigh activity in the range of pH 6-10 or 7-11 with linoleic acid orlinolenic acid as substrate.

The stability of the enzyme was determined after 1 hour incubation at40° C. at various pH values. The enzyme was found to have good stabilityin the pH range 4-10.

Example 9 Substrate Specificity of Lipoxygenase

The activity of lipoxygenase produced as in Example 4 was tested onvarious substrates as described above. The results are expressed ask_(cat) (or V_(max)), K_(M) and k_(cat)/K_(M) according to theMichaelis-Menten equation:

k_(cat) K_(M) Substrate micro-mol/min/mg mM k_(cat)/K_(M) Linoleic acid5.63 0.0068 828 Arachidonic acid 0.296 0.0175 16.9 Linoleyl alcohol 3.320.0034 982 Methyl linoleate 1.37 0.164  8.39 Monolinolein 85.41,3-dilinolein 12.4 Trilinolein 9.15

The lipoxygenase showed about twice as high activity toward linolenicacid than linoleic acid at pH 7.

Example 10 Bleaching of β-Carotene by Native Mn-Lipoxygenas

Purified Mn-lipoxygenase was used to bleach beta-carotene at pH 4.5, 6.5and 9.5. The highest bleaching activity was found at pH 6.5.

Example 11 Effect of LAS on Mn-Lipoxygenase

The activity of G. graminis lipoxygenase produced as in Example 4 wasmeasured with LAS up to 400 ppm at pH 7.0 and pH 10. The lipoxygenasewas found to be fully stable against LAS up to 400 ppm (the highestconcentration tested) at pH 7 and 10. This indicates that thelipoxygenase is stable enough at normal washing conditions, typically pH10 with 200 ppm of LAS.

1. An isolated polypeptide having lipoxygenase activity which: a) has atleast 98% identity with the mature polypeptide of SEQ ID NO: 2; or b) isencoded by the lipoxygenase-encoding part of the DNA sequence clonedinto a plasmid present in Escherichia coil deposit number DSM
 13586. 2.The polypeptide of claim 1, wherein the polypeptide has lipoxygenaseactivity and is encoded by the lipoxygenase-encoding part of the DNAsequence cloned into a plasmid present in Escherichia coil depositnumber DSM
 13586. 3. A dough composition comprising a polypeptide ofclaim
 1. 4. A detergent composition comprising a polypeptide of claim 1and a surfactant.
 5. The detergent composition of claim 4, wherein thesurfactant is an anionic surfactant.
 6. The detergent composition ofclaim 4, wherein the surfactant is a linear alkyl benzenesulfonate. 7.The isolated polypeptide of claim 1, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO:2.
 8. The isolated polypeptide ofclaim 1, wherein the polypeptide consists of the amino acid sequence ofSEQ ID NO:2.
 9. An isolated polypeptide having lipoxygenase activitywhich is encoded by the lipoxygenase-encoding part of the DNA sequencecloned into a plasmid present in Escherichia coli deposit number DSM13586.
 10. The isolated polypeptide of claim 9, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:2.
 11. The isolatedpolypeptide of claim 9, wherein the polypeptide consists of the aminoacid sequence of SEQ ID NO:2.
 12. A dough composition comprising thepolypeptide of claim
 9. 13. A detergent composition comprising thepolypeptide of claim 9 and a surfactant.